The long-term goal of this project is to define general principles and molecular mechanisms of Aquaporin (AQP) integration, folding and tetrameric assembly in the endoplasmic reticulum (ER) membrane. Aquaporins comprise a conserved family of 6-spanning, homotetrameric membrane proteins that play critical roles in water homeostasis in the kidney, lung, brain and other tissues. The molecular basis of water transport is achieved by the precise arrangement of six transmembrane helices and two half helices in a two-fold inverted symmetry around a monomeric pore. This structure is generated by the coordinated actions of the ribosome and Sec61 translocation machinery. A major unanswered question in this field is how subtle changes in nascent peptide structure influence this machinery to direct unique, and often pathological folding events. Interestingly, closely related AQPs exhibit different native folding pathways that are specified by just three variant residues. In addition, inherited point mutations in AQP2 disrupt folding and thereby cause nephrogenic diabetes insipidus (NDI), a life threatening disease of impaired urinary concentration. AQPs are therefore ideal model substrates for investigating normal and pathological mechanisms of membrane protein biogenesis that are implicated in a growing number of human protein folding disorders. The current proposal will use modified aminoacyl tRNAs to cotranslationally insert photocrosslinking and fluorescent probes into nascent AQP integration intermediates that are kinetically trapped at defined stages of synthesis. This approach provide a powerful new method to determine how the ER translocation machinery coordinates nascent chain folding in lumenal, cytosolic and membrane environments that closely mimic conditions in the cell. With these techniques we will: 1) define the molecular basis responsible for different AQP folding pathways, 2) define precisely how AQP2 folding is disrupted in nephrogenic diabetes insipidus, 3) define the functional and structural basis of AQP tetramerization required for intracellular trafficking. Results of these studies will significantly advance our understanding of AQP biology and improve our general ability to understand folding properties of complex integral membrane proteins. They will also establish a useful platform to investigate how folding is corrupted by inherited mutations, and thereby ultimately facilitate new strategies to treat diverse protein- folding disorders. PUBLIC HEALTH RELEVANCE: Disorders of membrane protein folding represent a rapidly expanding area of medicine that affects tens of thousands of Americans at enormous economic and social cost. Treatments for these disorders have been limited because basic understanding of biological folding pathways remains largely unknown for membrane proteins. This project will use novel biophysical approaches to define when transmembrane segments begin to fold in the context of ER biosynthetic machinery, how they are inserted into the ER membrane, and specific steps at which folding is disrupted by inherited disease- related mutations.